Sub cloning

In article <33e6a7df.15174351 at news.erols.com>, hadi at hatespam.helix.nih.gov
(hadi al-hasani) wrote:
> Hi Lisa,
> I have had the same problem with an insert which was toxic for the
> bugs when cloned in one direction into the Bluescript KS - but not in
> Bluescript SK...
> I suppose you need to know if indeed your insert is toxic. (If it's
> toxic you'll ligate until the end of time...)
>> Try the following:
> Make primers upstream and downstream of your MCS, respectively (or use
> your sequencing primers if you have any) and run a PCR with your
> ligation reaction. If you get your 4.5 kb product you definitely loose
> your ligation products. If you don't see any "insert" band something
> is wrong with your ligation. Don't forget to include a control to see
> if you can amplify xxx kb with our primers.
If the vector is dephosphorylated the product of the ligation will be
nicked, and you wont be able to amplify it whether or not it is there!
Martin
(mjakt at hkusua.hku.hk)